Establishment of a potency test by ELISA for a rabies vaccine for animal use in Japan

The ELISA we developed was able to determine the antigen content and was suitable for a potency test, and we described a relative potency assay method which determines the potency of test vaccines by comparing the ELISA value of a test vaccine to that of a reference vaccine. In the present study, we standardized the reference vaccine used for determining the potencies of test vaccines, and established a potency test by ELISA. We evaluated the proposed reference vaccine by the neutralizing antibody responses in dogs after vaccination, by the challenge protection test in guinea pigs (GP potency test), which is the earlier official potency test used in Japan, and by the NIH potency test, which is widely used throughout the world. The results showed that a 4-fold dilution of the proposed reference vaccine induced sufficient immunity in dogs. A 3-fold dilution of the proposed reference vaccine passed the GP potency test. The international units (IU) calibrated by the NIH potency test were 3.7 IU/dose. From the results and the WHO recommendation that veterinary rabies vaccines should have a potency of at least 1.0 IU/dose, we determined to dilute the proposed reference vaccine by 3 fold and regarded it as the reference vaccine. Finally, we confirmed that there is a good agreement between the results of the potency test by ELISA and the results of the GP potency test. The establishment of the potency test by ELISA has made it possible to monitor the potency in the production process and has contributed to the stable production of the vaccine.     

Quantitative analysis of microfilarial periodicity of Dirofilaria immitis in cats

Microfilarial periodicity of Dirofilaria immitis in the venous blood of infected cats was analyzed by a trigonometric model. Cats were infected by subcutaneous transplantation with 120-day-old juvenile D. immitis. Microfilariae in the blood were first observed 98 days after transplantation. Blood was collected at 4h intervals for a 24h period, and examinations were repeated five times in two cats. The calculated periodicity index was 75.1 and 50.3 in these two cats. The estimated hour of peak microfilarial density ranged from 1.00 to 2.84h. Thus, the periodicity of microfilariae of D. immitis in the blood of cats was characterized as nocturnally sub-periodic.     

Gonadotropin‐induced Puberty Does Not Impair Reproductive Performance of Gilts over Three Parities

The aim of this study was to evaluate the reproductive performance of three parities of gilts treated or not treated with gonadotropin to induce puberty. Sixty gilts received 600 IU of equine chorionic gonadotropin (eCG) followed by 2.5 mg of porcine luteinizing hormone (LH) 72 h later. Fifty‐nine other gilts were exposed only to a mature boar for 15 min twice daily. Artificial insemination (AI) was performed at 0, 12 and 24 h after the detection of oestrus, and gestation was confirmed by ultrasound after 35 days. Sows were inseminated at the first post‐weaning oestrus. The total numbers of piglets born, piglets born alive, stillborn, mummified foetuses, as well as pregnancy and farrowing rates were evaluated for each of the three parities. Culling rates, farrowing intervals and weaning‐to‐oestrous intervals (WEI) were also analysed. Mean age at puberty and oestrous manifestation were not significantly different between treatments (p = 0.0639; 179.20 ± 17.52 compared with 173.96 ± 16.94, 91.66% compared with 94.92%) across the experimental period. However, females that underwent puberty induction showed modest increases both in the number of total pigs born and in the number of piglets born alive. In conclusion, puberty induction through exogenous gonadotropin administration in field conditions did not induce a more concentrated first oestrous manifestation, but trended to a modest increase in the number of pigs born alive in the first parity and a reduced culling rate during the first gestation.     

Alteration of stimulus-specific guard cell calcium oscillations and stomatal closing in Arabidopsis det3 mutant

Cytosolic calcium oscillations control signaling in animal cells, whereas in plants their importance remains largely unknown. In wild-type Arabidopsis guard cells abscisic acid, oxidative stress, cold, and external calcium elicited cytosolic calcium oscillations of differing amplitudes and frequencies and induced stomatal closure. In guard cells of the V-ATPase mutant det3, external calcium and oxidative stress elicited prolonged calcium increases, which did not oscillate, and stomatal closure was abolished. Conversely, cold and abscisic acid elicited calcium oscillations in det3, and stomatal closure occurred normally. Moreover, in det3 guard cells, experimentally imposing external calcium-induced oscillations rescued stomatal closure. These data provide genetic evidence that stimulus-specific calcium oscillations are necessary for stomatal closure.     

Original Study: Comparison of three intraosseous access techniques in cats

Objective – To compare the placement feasibility and amount of bone trauma induced by 3 intraosseous (IO) access techniques in cats: an automatic impact penetration device (A), an automatic rotary insertion device (B), and a manual IO needle (C). Design – Prospective ex vivo study. Setting – University. Animals – Eighteen adult mixed breed feline cadavers. Interventions – Cadavers provided 72 total IO insertion locations divided equally between the right and left humerus and tibia. The 3 IO techniques were randomly allocated to these locations. Time to successful insertion, ease of insertion, and success rate were recorded. Each insertion site was analyzed for the number of bone fragments and defect diameter by computed tomography. Measurements and Main Results – Device B had lower time of insertion (P=0.01) compared with devices A and C. Device B had better ease of insertion scores (P<0.01) compared with devices A and C. No differences were detected between insertion sites (tibia versus humerus). No differences in the number of bone fragments, defect diameter, or success rate were detected among devices (P=0.06, 0.31, and 0.14, respectively). Conclusions – All 3 IO access methods evaluated yield acceptable results. Device B is significantly faster and easier to place in cat cadavers when compared with other methods.     

Oesophageal compliance in naturally occurring canine megaoesophagus

SUMMARY The passive blomechanical property of oesophageal compliance (OC) was measured in 8 naturally occurring cases of canine megaoesophagus, 8 matched control and 7 vagotomised control dogs. Of the 8 dogs with megaoesophagus, 6 had congenital Idiopathic megaoesophagus and 2 had secondary megaoesophagus attributable to generalised skeletal muscle disease. Stepwise distension of the whole oesophagus was employed for measurement of OC at the 4.0 and 8.0 mL/kg Injected volume steps within the control volume range (0 to 12.0 mL/kg). At both Injected volume steps OC was higher In megaoesophagus dogs than in either matched control or vagotomised control dogs (P / 0.01 In both cases), and no significant difference was observed In OC between matched control and vagotomised control dogs. No correlation was demonstrated between OC and the estimated duration of clinical signs of dogs with megaoesophagus. These findings suggest that In most cases of canine megaoesophagus the viscoelastic properties of the oesophageal wall are significantly altered, that In such cases the disorder is unlikely to be purely dynamic and that processes other than the duration of oesophageal dilatation are responsible for the alteration in oesophageal wall blomechanical properties. The relevance of these findings to current concepts on pathophysiological mechanisms underlying the evolution and resolution of various forms of canine megaoesophagus Is discussed.     

Susceptibilities against bovine lactoferrin with microorganisms isolated from mastitic milk

Antibacterial effects of bovine lactoferrin were studied in vitro against microorganisms isolated from mastitic milk in Tokachi area, Hokkaido, Japan. Microorganisms isolated were Escherichia coli (11 isolates), Klebsiella pneumoniae (5 isolates), enterococci (8 isolates), Staphylococcus aureus (10 isolates), coagulase negative staphylococci (CNS, 13 isolates), streptococci (11 isolates), Prototheca zopfii (7 isolates) and yeast-like fungi (9 isolates). Lactoferrin has been known as a multifunctional protein and its antimicrobial effect is one of the most essential function of it. In order to compare their susceptibilities against lactoferrin, the minimal inhibitory concentration values were estimated by a microplate assay method using 96-well microplate, which involved measuring the optical density of the cultures. Prototheca zopfii was highly sensitive to bovine lactoferrin and complete inhibition of this microorganism was observed even at the low concentration of 7 mug/ml. On the other hand, E. coli and enterococci showed resistance against lactoferrin action and staphylococci showed strain-dependent resistance.     

Abnormal functions of Leydig cells in crossbred cattle–yak showing infertility

In Mongolia, yak (Bos grunniens) are able to live in alpine areas and their products greatly influence the lives of the local people. Increased vigour in hybridized yak and cattle can offer benefits for livestock farmers. However, male hybrids show reproductive defects resulting from spermatogenesis arrest, affecting the conservation and maintenance of dominant traits in the next generation. The underlying mechanisms involved in hybrid cattle–yak infertility have recently been investigated; however, the genetic cause is still unclear. Androgens and androgen receptor (AR) signalling are required for spermatogenesis. We, therefore, evaluated the expression of AR, 3β-hydroxysteroid dehydrogenase (3βHSD) and 5α-reductase 2 (SRD5A2) in Leydig cells to investigate their function in cattle–yak spermatogenesis. Testicular tissues from yaks (1–3 years old) and hybrids (F1–F3, 2 years old) were collected and subjected to immunohistochemistry and image analyses to investigate the expression of each parameter in the Leydig cells. After maturation at 2 years, the expression levels of AR increased and the levels of 3βHSD decreased, but the SRD5A2 levels remained constant in yak. However, the cattle–yak hybrid F2 showed immature testicular development and significantly different expression levels of AR and 3βHSD compared with mature yak. These results suggest that the decreased expression of AR and increased expression of 3βHSD in the Leydig cells of cattle–yak hybrid testes may represent one of the causes of infertility. Our study might help in solving the problem of infertility in crossbreeding.